The present invention relates generally to arrays of immunoglobulin binding proteins. The invention is more particularly related to methods for the expression of arrays of foreign immunoglobulin binding proteins in eukaryotic cells, such as plant cells, as well as to transformed eukaryotic cells that express such arrays.
Immunoglobulin molecules play key roles in a variety of physiological processes. Such molecules, which include antibodies and portions thereof, are critical for immune system function, and have found numerous therapeutic and diagnostic applications. The discovery of immunoglobulin molecules with desired binding characteristics is the focus of many current drug discovery efforts.
Traditional techniques for immunoglobulin molecule discovery involve the expression of a multitude of immunoglobulin molecule genes in an array of hybridoma cells, other forms of immortalized B-lymphocytes or phage-infected bacteria. For monoclonal antibody expression, individual antibody-producing B-lymphocytes from an immunized animal are generally fused with cells derived from an immortalized B-lymphocyte tumor. Clones of hybrid cells are then screened to identify those that grow indefinitely and secrete the desired immunoglobulin molecule. The polynucleotides encoding the monoclonal antibodies can then be isolated and used to express all or part of the antibody in other organisms, such as bacteria, yeast and plants. The ability to express immunoglobulins on the surface of bacteriophage has enabled the generation of immunoglobulin libraries that could represent all possible combinations of heavy and light chains derived from any population of B-lymphocytes. These libraries have been used successfully to identify high affinity combining sites recognizing a wide variety of antigens. A significant drawback to this technique is the randomization of heavy and light chain combining sites requiring the generation of very large numbers of recombinant phage to identify specific heavy and light chain binding pairs. This combinatorial aspect of random libraries makes expression of these libraries in other organisms unfeasible. Newer technologies involve transgenic mice expressing antibodies from human chromosomal segments which can be used to generate hybridoma arrays expressing human antibodies.
Arrays formed in B-lymphocytes, phage infected bacteria or transgenic animals have been useful within certain immunoglobulin molecule screens, but difficulties have been encountered with producing large quantities of immunoglobulin molecules in these cells. Large-scale production of immunoglobulin molecules from any of the traditional organisms is typically very expensive. Further, phage infected bacteria are incapable of providing the variety of immunoglobulin molecule structures that may be desired. Similarly, the usefulness of transgenic animal cells has been limited by the susceptibility of such cells to infection with viruses or other microorganisms.
For economic and other reasons, it would be desirable to use genetically engineered plants as the primary vehicle for the discovery of immunoglobulin molecules, as well as for the ultimate production of immunoglobulin molecules to be used in industrial, clinical or research applications. The advantages of plants for production of immunoglobulin molecules include a low cost of production, relatively low capital investment compared to fermentation systems, the absence of animal viruses and prions, production of the immunoglobulin molecule in a biochemical background of defined proteins such as seed proteins, ease of storage and transport, and a facile scale-up to unlimited quantities of raw material. It would also be desirable to be able to express a library of binding proteins that is not derived from a combinatorial process of randomly paired heavy and light chains.
It is known that immunoglobulin molecules can be expressed in a variety of eukaryotic hosts including plant cells. A wide variety of structural genes have been isolated from mammalian cells and viruses, joined to transcriptional and translational initiation and termination regulatory signals from a source other than the structural gene, and introduced into plant hosts in which these regulatory signals are functional. Among those host cells that have been transformed with individual immunoglobulin molecule-encoding nucleic acids are monocots (e.g., corn, rice and wheat), dicots (e.g., tobacco, soybean, alfalfa, petunia, and Arabidopsis) and lower plants (e.g., Chlamydomonas). Plants transformed with nucleic acids encoding individual immunoglobulin molecules have been able to produce fully functional and fully assembled immunoglobulins (see Hiatt et al., Nature 342:76-78, 1989; Firek et al., Plant Molecular Biology 23:861-870, 1993; Van Engelen et al., Plant Molecular Biology 26:1701-1710, 1994; Ma et al., Science 268:716-719, 1995; Magnuson et al., Protein Expression and Purification 7:220-228, 1996; Schouten et al., Plant Molecular Biology 30:781-793, 1996; Fiedler et al., Immunotechnology 3:205-216, 1997; Verch et al., J. Immunol. Meth. 220:69-75, 1998; Zeitlin et al., Nature Biotechnology 16:1361-1364, 1998; DeJaeger et al., Eur. J. Biochem. 259:426-434, 1999; Fischer et al., Biol. Chem. 380:825-839, 1999; Khoudi et al., Biotechnology and Bioengineering 64(2):135-143, 1999; McCormick et al., Proc. Natl. Acad. Sci. USA 96:703-708, 1999; Russell, Curr. Top. Microbiol. Immunol. 240:119-138, 1999).
In previous plant cell transformations, the transforming nucleic acid introduced a single immunoglobulin molecule. For example, tobacco plants have been transformed with individual gamma or kappa chains to produce individual plants expressing immunoglobulin molecule components. The respective tobacco transformants were then cross-pollinated to produce plants expressing a single antibody, wherein covalent bond formation between the two components resulted in the formation of enhanced binding capacity. In another instance, an antibody molecule was introduced into a single plant using a single vector. The vector encoded two immunoglobulin component chains and resulted in the formation, in the plant, of an immunoglobulin molecule comprising covalently linked heavy and light immunoglobulin chains.
Plant cells have not been used to express a diversity of immunoglobulin molecules in an array. As noted above, the ability to prepare an array of immunoglobulin molecules in plants or plant cells would facilitate identification of useful immunoglobulin molecules and would enable a rapid transition from immunoglobulin molecule discovery to full scale production in a single organism.
Accordingly, there remains a need in the art for methods for generating arrays of immunoglobulin molecules in plants and plant cells, as well as other eukaryotic organisms and cells. The present invention fulfills these needs and further provides other related advantages.
Briefly stated, the present invention provides methods for the production of arrays of biologically or physiologically active immunoglobulin binding proteins in eukaryotic cells. Within certain aspects, methods are provided for preparing an immunoglobulin binding protein array in plant cells, comprising the steps of: (a) transforming a population of plant cells with a library of at least two different polynucleotides encoding different immunoglobulin binding protein (IgBP) polypeptides that: (i) specifically bind to a ligand with a KD less than 10xe2x88x926 moles/liter; or (ii) form one or more disulfide bonds with one or more polypeptides in the transfected cell, to generate a binding protein that specifically binds to a ligand with a KD less than 10xe2x88x926 moles/liter; wherein the IgBP polypeptides (i) comprise four framework regions (e.g., human or murine) alternating with three complementarity determining regions and (ii) comprise at least one peptide sequence having at least 75%, preferably at least 95%, sequence identity to a framework region of a native IgM, IgG, IgA, IgD, IgE, IgY, kappa or lambda immunoglobulin molecule; and wherein the IgBP polypeptides are not detectably expressed by the plant cells prior to transformation; and (b) selecting transformed plant cells, and therefrom preparing an IgBP array in plant cells. Each IgBP polypeptide may be a functional IgBP or an IgBP component (e.g., a portion of an immunoglobulin molecule selected from the group consisting of heavy chains and fragments thereof, light chains and fragments thereof, J chains and secretory components) that, upon disulfide linkage to one or more IgBP components encoded by other polynucleotides in the library, forms a functional IgBP. Within certain specific embodiments, a library employed in such methods commprises at least 10, 100, 1,000 or 10,000 different polynucleotides.
Within certain embodiments, such methods further comprise the step of: (c) growing the transformed plant cells on a growth medium that supports replication of the plant cells, such that functional IgBPs are assembled by the plant cells. Within other specific embodiments, such methods further comprise the steps of: (c) growing the transformed plant cells on a growth medium to form plants; and (d) sexually crossing the plants with themselves or other plants to generate progeny, such that the progeny comprise polynucleotides encoding IgBP components sufficient to form a functional IgBP. Such progeny may be seeds, or may be plants or plant cells that assemble functional IgBPs, and the IgBP polypeptides may, but need not, be secreted from the plant cells.
The present invention further provides, within other aspects, methods for preparing a heavy chain binding protein array in eukaryotic cells (e.g., plant, insect or mammalian cells), comprising the steps of: (a) transforming a population of eukaryotic cells with a library of at least two different polynucleotides, wherein each polynucleotide encodes a different heavy chain binding protein (CHBP) polypeptide that: (i) comprises an amino acid sequence that is at least 75% identical to a constant region tailpiece of a mu or alpha chain of a native immunoglobulin heavy chain; (ii) comprises multiple combining sites, wherein all of the combining sites satisfy the same one of the following requirements: (1) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin light chain variable region or (2) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin heavy chain variable region; and (iii) either (1) specifically binds to a ligand with a KD less than 10xe2x88x926 moles/liter; or (2) forms one or more disulfide bonds with one or more polypeptides in the transfected cell, to generate a CHBP that specifically binds to a ligand with a KD less than 10xe2x88x926 moles/liter; and (b) growing the transformed cells on a medium that permits assembly of CHBPS, wherein each CHBP comprises at least four combining sites; and therefrom preparing a CHBP array in eukaryotic cells. The polynucleotides may, for example, encode immunoglobulin alpha or mu chains. Within certain embodiments, the cells are further transformed with one or more polynucleotides encoding polypeptides having sequences that are at least 75% identical to a sequence of an immunoglobulin J chain. Resulting CHBPs may be assembled, for example, from four alpha chains and one J chain, from twelve mu chains and/or from ten mu chain and at least one J chain. CHBPs or components thereof may, but need not, further comprise one or more portions of immunoglobulin molecules selected from the group consisting of J chains, secretory components and light chain constant regions. The CHBPs may accumulate in an intracellular compartment of the cells or may be secreted from the cells.
Within further aspects, methods are provided for preparing a heavy chain binding protein array in eukaryotic cells, comprising the steps of: (a) exposing multiple copies of a polynucleotide encoding a native heavy chain to a mutagen, such that random or site-directed mutagenesis of the polynucleotide occurs, resulting in a library of heavy chain variants; (b) transforming a population of eukaryotic cells with the library of heavy chain variants; and (c) growing the transformed cells on a medium that permits assembly of CHBPS, wherein each CHBP comprises at least four combining sites; and therefrom preparing a CHBP array in eukaryotic cells.
Methods are further provided for preparing a plant CHBP array, comprising the steps of: (a) transforming a population of plant cells with a library of at least two different polynucleotides, wherein each polynucleotide encodes a different CHBP component that forms one or more disulfide bonds with one or more polypeptides in the transformed cell to generate a CHBP that specifically binds to a ligand with a KD less than 10xe2x88x926 moles/liter, wherein each component: (i) comprises an amino acid sequence that is at least 75%, preferably at least 95%, identical to a constant region tailpiece of a mu or alpha chain of a native immunoglobulin heavy chain; and (ii) comprises multiple combining sites, wherein all of the combining sites satisfy the same one of the following requirements: (1) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin light chain variable region or (2) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin heavy chain variable region; (b) growing the transformed plant cells on a growth medium to form plants; and (c) sexually crossing the plants to generate progeny, such that the progeny comprise polynucleotides encoding CHBP components sufficient to form a functional CHBP that comprises at least four combining sites; and therefrom preparing a plant CHBP array. The progeny may be seeds, or may be plants or plant cells that assemble functional CHBPS. Within certain specific embodiments, a library employed in such methods commprises at least 10, 100, 1,000 or 10,000 different polynucleotides. The CHBPs may accumulate in an intracellular compartment of the cells or may be secreted from the cells.
Within further aspects, the present invention provides CHBP arrays in eukaryotic cells, comprising at least two eukaryotic cells (e.g., plant, insect or mammalian cells) that are each transformed with a different polynucleotide encoding at least one CHBP polypeptide that: (a) comprises an amino acid sequence that is at least 75% identical to a constant region tailpiece of a mu or alpha chain of a native immunoglobulin heavy chain; (b) comprises multiple combining sites, wherein all of the combining sites satisfy the same one of the following requirements: (i) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin light chain variable region or (i) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin heavy chain variable region; (c) either (i) specifically binds to a ligand with a KD less than 10xe2x88x926 moles/liter; or (ii) forms one or more covalent bonds with one or more polypeptides in the transfected cell, to generate a CHBP that specifically binds to a ligand with a KD less than 10xe2x88x926 moles/liter; and (d) differs in amino acid sequence from other CHBPs in the array; wherein the cells assemble CHBPs comprising at least four combining sites. The polynucleotides may, for example, encode polypeptide components of immunoglobulin molecules independently selected from the group consisting of heavy chains and fragments thereof, light chains and fragments thereof, J chains and secretory components. Within certain specific embodiments, the cells in the array assemble at least 10, 100, 1,000 or 10,000 different polynucleotides. Also within certain embodiments, each cell in such an array may be transfected with at least two different polynucleotides, each encoding a different CHBP component, such that each cell assembles a functional CHBP comprising the CHBP components.
Within further aspects, the present invention provides compositions comprising an array of encapsulated CHBPS, wherein each CHBP: (a) comprises an amino acid sequence that is at least 75% identical to a constant region tailpiece of a mu or alpha chain of a native immunoglobulin heavy chain; (b) comprises at least four combining sites, wherein all of the combining sites satisfy the same one of the following requirements: (i) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin light chain variable region; or (ii) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin heavy chain variable region; and (c) either (i) specifically binds to a ligand with a KD less than 10xe2x88x926 moles/liter; or (ii) forms one or more covalent bonds with one or more polypeptides in a cell, to generate a CHBP that specifically binds to a ligand with a KD less than 10xe2x88x926 moles/liter; and (d) differs in amino acid sequence from other CHBPs in the array.
Methods are further provided for preparing a heavy chain binding protein array in eukaryotic cells, comprising the steps of: (a) exposing multiple copies of a polynucleotide encoding a native heavy chain to a mutagen, such that random or site-directed mutagenesis of the polynucleotide occurs, resulting in a library of heavy chain variants; (b) transforming a population of eukaryotic cells with the library of heavy chain variants; and (c) growing the transformed cells on a medium that permits assembly of CHBPS, wherein each CHBP comprises at least four combining sites; and therefrom preparing a CHBP array in eukaryotic cells.
Within further aspects, the present invention provides CHBPs that: (a) comprise an amino acid sequence that is at least 75% identical to a constant region tailpiece of a mu or alpha chain of a native immunoglobulin heavy chain; (b) comprise at least four combining sites, wherein all of the combining sites satisfy the same one of the following requirements: (i) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin light chain variable region; or (ii) at least 75% identity to a 25 consecutive amino acid portion of an immunoglobulin heavy chain variable region; and (c) either (i) specifically bind to a ligand with a KD less than 10xe2x88x926 moles/liter; or (ii) form one or more covalent bonds with one or more polypeptides in a cell, to generate a CHBP that specifically binds to a ligand with a KD less than 10xe2x88x926 moles/liter.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.